Despite considerable efforts for close to thirty years, we are still far from having an effective vaccine against HIV-1. Recent results of the RV144 efficacy trial have provided the first proof in humans that HIV infection can be diminished by vaccination and showed that the antibody response against the HIV-1 surface protein Env, contributes significantly to protection. A major hurdle to an HIV-1 vaccine is the availability of n immunogen that elicits broadly neutralizing antibodies. Lack of high resolution structural information for native, unliganded Env as well as its instability, flexibility, glycosylation and hgh sequence diversity are major contributors to this problem. An important recent development is the availability of medium resolution structures of a part of the Env ectodomain that reveal some details of gp120:gp41 interactions. However these lack some functionally important regions of the ectodomain, sidechain locations for the gp41 subunit, possess an additional disulfide bond and are complexed to antibody ligands. While some of these limitations may be overcome in future studies, it is unlikely that there will be a high resolution crystal structure of functional unliganded Env in the context of intact virus in the near future. The goals of this proposal are to apply saturation suppressor mutagenesis methodology in conjunction with appropriate infectivity screens, deep sequencing and protein labeling to obtain insight into the structure, stability and dynamics of intact Env in its native context on the viral surface. There are three specific Aims. I Aim 1 we will use saturation and saturation-suppressor mutagenesis to validate and extend structural information recently derived from gp140 trimers to full length Env on virions, and to identify trimer stabilizing mutations. Increased structural information is vital to understanding Env function and the mechanism of viral entry. Improving the stability and rigidity of unliganded trimers should improve the elicitation of neutralizing antibodies when such molecules or their derivatives are used as immunogens. In Aim2 we will probe Env dynamics using Cys labeling of env on intact virions. Little is known at the molecular level about the flexibility and stability o the native HIV-1 trimer on virions. Such information is also vital for designing better immunogens and for understanding viral entry. In Aim3 we will design fragment models of the gp120:gp41 interface based on structural input from existing structures and mutagenesis data. Stabilizing mutations derived from these studies will be incorporated into soluble gp120:gp41 complexes, which are potential immunogens. These simplified interface models will also be used to design inhibitors of viral entry.